PROJECT SUMMARY Cellular senescence is a bona fide tumor suppression mechanism but also a cause of cell and tissue aging. Senescence is caused by a range of cellular stresses and characterized by an irreversible proliferation arrest and a potent pro-inflammatory phenotype, the senescence-associated secretory phenotype (SASP). Senescence-associated proliferation arrest and SASP cooperate in tumor suppression, by arresting proliferation of damaged pre-malignant cells and promoting immune clearance of the damaged cells. However, over the longer term, as a source of chronic inflammation, SASP also promotes tissue aging and disease. Consequently, there is currently much effort devoted to development of pharmacologic approaches to eliminate senescent cells to promote healthy aging. However, these so-called senolytic drugs tend to show unwanted toxicities. An alternative, inherently less toxic approach, is to specifically inhibit the pro-aging chronic SASP. Evidence indicates that this can be achieved without impairing tumor suppressive senescence-associated proliferation arrest. Towards an understanding of SASP and targets for its inhibition, we recently showed that senescent cells shed fragments of nuclear chromatin into the cytoplasm, so-called cytoplasmic chromatin fragments (CCF), via a nucleus-to-cytoplasmic blebbing process. CCF initiate SASP in senescent cells, via activation of the anti-viral cytoplasmic DNA sensing apparatus to activate NFkB and hence SASP. Preliminary data indicate that formation of CCF is triggered by dysfunctional mitochondria which signal to the nucleus via a retrograde mitochondrial ROS-JNK kinase-53BP1 pathway to nuclear MRE11 exonuclease, whose activity is required for CCF formation. Accordingly, inhibitors of mitochondrial ROS, JNK, MRE11 and HDAC inhibitors (which appear to act indirectly by improving mitochondria function) all suppress CCF and SASP. To identify additional tool compounds to probe the mechanism of CCF formation and/or approved drugs as candidates for repurposing as anti-SASP/pro-healthy aging interventions, we have established an automated high content screen for inhibitors of CCF. We will screen focused collections of mechanistically well-defined small molecules and drug repurposing libraries to identify and validate novel inhibitors of CCF and SASP. Completion of these Specific Aims will identify valuable tool compounds for further mechanistic investigation of CCF and SASP, and also identify lead compounds for repurposing as anti-SASP/pro-healthy aging interventions in humans.